EP1399727B1 - Dispositifs de colonnes de filtration et procedes de filtration associes - Google Patents

Dispositifs de colonnes de filtration et procedes de filtration associes Download PDF

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Publication number
EP1399727B1
EP1399727B1 EP02742051.2A EP02742051A EP1399727B1 EP 1399727 B1 EP1399727 B1 EP 1399727B1 EP 02742051 A EP02742051 A EP 02742051A EP 1399727 B1 EP1399727 B1 EP 1399727B1
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EP
European Patent Office
Prior art keywords
filter column
filter
body portion
collection tube
column
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EP02742051.2A
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German (de)
English (en)
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EP1399727A2 (fr
Inventor
Derrick A. Richardson
Derek S. Pai
Jorge Monteon
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Life Technologies Corp
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Life Technologies Corp
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/042—Caps; Plugs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/043—Hinged closures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption

Definitions

  • This invention relates to filtration filter columns and methods of filtering therewith. More specifically, this invention relates to system and methods for isolating nucleic acids such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) from other materials such as enzymes, salts, buffers, small molecules, and cellular debris.
  • nucleic acids such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • a known method for isolating nucleic acids from a small amount of starting material includes the use of a spin filter column ("filter column") that contains a nucleic acid binding material (i.e., a filter).
  • a nucleic acid binding material i.e., a filter
  • binding material/filters include silicas like glass powder, silica particles, glass microfibers, diatomaceous earth, etc. These filters are often associated with a "filter surface area.” This filter surface area is not limited to the surface area of a side of the filter. Instead, since the filters are usually comprised of microscopic fibers, particles, porous substances, etc., the filter surface area is actually defined by the surface area of the components which comprise the filter.
  • a filter that comprises glass microfibers may have a filter surface area defined by the surface area of the microfibers within the filter (either all of the microfibers or a portion thereof).
  • filter columns may isolate nucleic acids directly from cells or biological tissue.
  • a filter column is inserted into a microcentrifuge tube (e.g., a 1.5 ml tube) and a solution containing nucleic acids along with undesirable impurities is loaded into the top of the filter column.
  • the starting material containing the nucleic acids is prepared from cells that have been treated with a disrupting solution causing the release of the nucleic acids.
  • the nucleic acid solution is the product of an earlier reaction step. In either case, the nucleic acid binds to the filter column filter in the presence of a chaotropic agent. Then the filter column is centrifuged in a microcentrifuge.
  • Centrifugation forces the solution through the filter column's filter and binds the nucleic acid to the filter.
  • the filter with the nucleic acids bound therein is washed by applying a washing solution to the top of the filter column, and centrifuging again. After each wash the filter column can be removed from the collection tube so that the collection tube may be emptied.
  • placement of an elution buffer usually water having a specific pH
  • centrifugation elutes the nucleic acid that is bound to the filter. Given the proper pH, the nucleic acid elutes into the liquid, and then collects into the collection tube.
  • the volumes of the binding and wash solutions can be relatively large, thus necessitating the use of a larger (1.5 - 2.0 mL) tube.
  • the volumes for elution are often smaller, making it desirable to have a smaller tube for the elution step. Eluting directly into a smaller tube allows one to proceed to the next reaction step in the small tube, rather than having to pipette out of the large tube.
  • use of a pipette is undesirable as it introduces the risk of loss of samples as well as contamination of the sample.
  • kits that include filter columns designed to use this technique for isolating nucleic acids.
  • QIAGEN, Promega, and Boehringer Mannheim GmbH offer filter columns based on the above described principle
  • existing filter columns cannot be used interchangeably with collection tubes of different sizes. Instead, these previously known filter columns only fit into a single size collection tube (e.g ., a standard 1.5 - 2.0 mL microcentrifuge tube).
  • An example of a filter device and kit for isolation and purification of nucleic acids and the like from heterogeneous biological samples is given in US 5,438,128 .
  • the device comprises a membrane assembly comprised of layers of microporous, polymeric membranes functionalized with ion-exchange groups, and is re-usable for like samples.
  • the remaining alternative is to use a filter column specifically designed to fit into a larger collection tube (e.g., a 1.5 mL - 2.0 microcentrifuge tube). While this alternative minimizes the additional wash steps and increased handling discussed above, the alternative presents additional problems. For instance, after purification, the nucleic acid solution must be eluted from the filter column into an appropriately sized 1.5 mL - 2.0 mL microcentrifuge tube. As discussed above, many applications may benefit by storing the elution buffer and nucleic acid in a smaller sized microcentrifuge tube.
  • the elution buffer and nucleic acid must then be transferred ( e.g., by aspiration) into a smaller (e.g., 0.5 mL) tube. Again, this extra transfer step introduces the undesirable potentials of contamination and loss of some of the nucleic acid.
  • US 4,214,993 describes a filter column (separating column) for use with a single size of tube.
  • the column forms part of an apparatus comprising three pieces which may be nested together to form a stacked array.
  • the three pieces are a cap, an extraction cartridge housing the column, and a primary recovery cup (tube).
  • a secondary recovery cup may also be nested onto the lower end of the primary cup.
  • Each piece has an enlarged upper portion forming an exterior step which facilitates mounting in the swinging bucket of a centrifuge.
  • WO 93/01494 A1 describes a method and apparatus for improved solid phase extraction, in particular for application in the field of drug screening.
  • the apparatus comprises a filter column for use with multiple sizes of centrifuge tubes. Portions of the filter column are sized to provide an interference fit with the rim of a standard centrifuge tube.
  • the invention described herein addresses the problems discussed above. Moreover, the invention described herein allows centrifugation from one filter column into at least two distinct sizes of collection tubes. The disclosed invention may be used with commercially available collection tubes.
  • the invention relates to filtration filter column devices and methods of filtering therewith, as defined in the appended claims.
  • the invention includes a filter column filter which has a plurality of bearing surfaces along with a plurality of body portions of varying outer diameters each of which is adapted to seat on respective collection tubes of different sizes.
  • a first aspect of the invention is directed to a filter column comprising: a body having a passageway extending therethrough, said body comprising at least a first body portion, a second body portion, and a third body portion, where an outer diameter of said first body portion is greater than an outer diameter of said second body portion, and where said outer diameter of said second body portion is greater than an outer diameter of said third body portion; a filter located within said passageway, said filter adapted to isolate nucleic acids from a liquid sample; a first bearing surface adapted to rest against the rim of a first collection tube where an outer diameter of the second body portion below the first bearing surface is adapted to fit securely with the first collection tube, wherein the first collection tube is a 1.5 - 2.0 mL centrifuge tube; and a second bearing surface adapted to rest against the rim of a second collection tube where an outer diameter of the third body portion below the second bearing surface is adapted to fit securely with the second collection tube and wherein the opening of the first collection tube is greater than the opening of the second
  • a preferred embodiment of the invention includes a lid which may be either discrete from the filter column or may be integral with the filter column body. In the latter case, the lid may be joined to the filter column body by a hinge or other member.
  • a preferred embodiment of the invention includes a filter column which contains vents within a passageway of the filter column.
  • the vents permit placement of a lid on the filter column without increasing the pressure within the passageway thereby causing sample material to be forced out of the filter column.
  • the filter column of the invention includes deformable ribs placed about an outer surface of a body portion of the filter column. Such deformable ribs may assist placement of the filter column within closely varying sizes of collection tubes.
  • the invention includes kits for isolation of nucleic acid from a solution containing nucleic acids and contaminating material.
  • the kit contains a filter column according to the first aspect, collection tubes, binding buffer, wash solutions and elution buffer.
  • a second aspect of the present invention is directed to a method for isolating a nucleic acid material comprising: providing a filter column according to the first aspect comprising: fitting the filter column into the first collection tube at the first bearing surface such that an outer diameter of the second body portion below the first bearing surface fits securely with the first collection tube; attaching a laser capture microdissection extraction device flange interface which seats a laser capture microdissection extraction device with biological material located on the bottom surface; transferring the solution containing nucleic acid to the filter of the filter column in the presence of an agent capable of promoting binding of the nucleic acid material to the filter; transferring the solution from the filter column to the first collection tube via centrifugal force; washing the filter column filter with a wash solution; removing the filter column from the first collection tube; providing a second collection tube wherein the opening of the second collection tube is smaller than the opening of the first collection tube; placing the filter column in the second collection tube at the second bearing surface such that an outer diameter of the third body portion below the second bearing surface fits securely with
  • Figure 1A shows a profile of a preferred embodiment in accordance with the present invention.
  • the filter column 1 includes a first body portion 3, a second body portion 5, and a third body portion 7.
  • the filter column 1 also contains a first bearing surface 9 and a second bearing surface 11 located between body portions 3, 5, 7 as illustrated.
  • the bearing surfaces are intended to permit placement of the filter column 1 in various collection tubes ( e.g ., see Figures 4 and 5 .) Accordingly, the present invention contemplates that the bearing surfaces, either alone, or along with body portion(s) adjacent to a bearing surface, serve to provide stability of a filter column within a collection tube.
  • bearing surfaces 9, 11 are located at the intersection of the respective body portions, in other variations of the invention, and within the scope of the appended claims, the bearing surfaces may be located anywhere along the various body portions. As illustrated in Figure 1B where the bearing surfaces 10, 12, are located along respective body portions of a filter column 1. Additionally, while bearing surfaces 9, 11, are illustrated as being tapered, the invention is not limited as such.
  • Preferred embodiments of the invention also include filter columns with more than three body portions.
  • the variation of the spin-column depicted in Figure 1 further includes an outer rim 17 adjacent to a top 15 of the filter column 1.
  • the outer rim 17 may also provide an additional bearing surface 19 and may also aid in grasping and manipulating the filter column 1.
  • the bottom of the filter column may be reduced in diameter 13 to assist in retaining a filter (not shown) within the filter column 1.
  • the body of the filter column may be adapted as required to accommodate any particular filtration process for example, centrifugation, vacuum filtering, or any known filtering process.
  • the filter column may also include a manifold to accommodate the vacuum.
  • Such modifications are well known to those familiar with filter columns and methods of using such devices.
  • the dimensions of the body portions 3, 7 are selected so that the filter column 1 may fit into various collection tubes that are sized for the respective body portion and bearing surface.
  • the invention include sizing of a filter column to include body portions and bearing surfaces that accommodate both a 1.5 - 2.0 mL and a 0.5 mL or a 0.2 mL microcentrifuge tube.
  • An example of such tubes includes PGC Scientifics No. 16-8105-52 (1.5 mL) supplied by PGC Scientifics Corporation of Maryland, Eppendorf No. 22 36 430-8 (0.5 mL) supplied by Eppendorf AG of Germany, and GeneAmp® No. N801-0611 (0.5 mL) supplied by Perkin-Elmer Life Science of Massachusetts.
  • the opening diameter for a 1.5 mL - 2.0 mL microcentrifuge tube may range from 8.1 - 9.4 mm (0.32 - 0.37 in).
  • the opening diameter for a 0.5 mL microcentrifuge tube may range from 6.6 - 6.9 mm (0.26 - 0.27 in).
  • the definition of diameter of the filter column may includes any such external feature as crush ribs, buttress, collar, or any other feature that may be present on a portion of a filter column body that is intended for insertion into a collection tube.
  • the filter column of the invention comprises deformable ribs as defined in appended claim 1.
  • the invention may be suited for use with the tubes described above, the invention is not limited to compatibility with such tubes.
  • embodiments of the inventive device described herein are discussed for use with existing microcentrifuge tubes ranging in size from 0.5 mL capacity to 1.5 - 1.5 mL capacity, the invention is not limited as such. Instead, within the scope of the appended claims, the inventive device may be applied to collection tubes as described herein, centrifuge tubes of any size, or any type of collection tube where a benefit from the improvements of the current invention are desirable.
  • the invention may be adapted to accommodate any number of combinations of large and small collection tubes, including, but not limited to a filter column adapted to accommodate a large 0.5 mL tube and a small 0.2 mL tube, or a filter column adapted to accommodate a large 1.5 mL tube and a small 0.2 mL tube.
  • the height of the filter columns of the present invention is selected so that the filter column along with the particular collection tube used will fit within the centrifuge apparatus (e.g ., an Eppendorf 5415C centrifuge supplied by Eppendorf AG of Germany.)
  • the centrifuge apparatus e.g ., an Eppendorf 5415C centrifuge supplied by Eppendorf AG of Germany.
  • the height which protrudes from the 0.5 mL microcentrifuge tube i.e., referring to Figure 1A , the height from the top of bearing surface 11 to the top of the device
  • the height from the top of bearing surface 11 to the top of the device must be below 15.9 mm (0.625 in) preferably below 12.7 mm (0.5) in.
  • the filter columns of the present invention may be fabricated from materials readily known to those familiar with existing filter columns. Such materials include, but are not limited to, polypropylene or polycarbonate. Polyethylene, Fluoropolymers such as polytetrafluoroethylene and polyvinylidine flouride, Polyarylene ether ketones, and co-polymers. It is often desirable to use a material which is thermoplastic to allow molding of the columns. The columns can also be machined out of appropriate materials. In some cases, it is desirable to choose materials which permit sterilization of the filter column thereby allowing the filter column, filter, and sample to be nuclease-free.
  • Figure 2 illustrates the internal body structure of a preferred embodiment of a filter column of the present invention.
  • Liquid solutions may be loaded into the top of the filter column 15.
  • liquid solutions include the nucleotide-containing solution, wash or rinsing solutions and an elution buffer (water or TE).
  • the nucleic acid solution can be from a lysate ( e.g . isolated directly from cells), or nucleic acids from a reaction mixture.
  • the nucleic acids from reaction mixtures could be from reactions such as PCR, DNA or RNA polymerization, reverse transcription, etc.
  • the nucleic acid solution Before being loaded onto the filter column the nucleic acid solution is usually combined with a binding buffer containing a chaotropic agent to aid in binding the nucleic acid to the filter.
  • compositions of binding buffers, wash or rinsing solutions and elution buffer is well known in the field and can be found in US patents 5,075,430 to Little , 5,808,041 to Padhye , and 5,652,141 to Henco .
  • the preferred embodiment of the invention depicted in Figure 2 also illustrates a vent 27.
  • the vent 27 may be located along an interior surface of a passageway 21 of a filter column 1 but will be placed in fluid communication with an exterior of the filter column 1.
  • the vent 27 permits venting of pressure within the passageway 21 during placement of a lid (not shown) in the top opening 15 of the filter column 1. Without a vent, the placement of a lid could increase pressure within the passageway 21 such that sample material is forced out of the bottom 13 of the filter column 1. Although such displacement of the material may not have an effect on the function of a device of the present invention, such an occurrence may be undesirable. It is contemplated that the device 1 may have any number of vents.
  • vents may be placed randomly or spaced evenly apart on a wall of a passageway. In one variation (not shown) four vents are placed at intervals of 90° along the walls of the passageway. It is further contemplated that a vent may be placed in another location in the filter column 1 or even within a lid (not shown) itself.
  • the preferred filter column 1 of the present invention also allows for varying placement of a filter (not shown) within a passageway 21 of the filter column 1.
  • a filter (not shown) within a passageway 21 of the filter column 1.
  • placement of a filter in a bottom 23 of the second body portion 5 permits the filter column 1 to provide a certain volume capacity.
  • the volume capacity of the filter column 1 can increase by changing the location of the filter to a bottom 25 of the third body portion 7. Accordingly, the same filter column 1 body may be used for varying application.
  • a filter can be located in the lowest chamber.
  • locating the filter towards a middle chamber reduces the volume of fluid that may be added but permit a larger filter surface area as the diameter of the middle chamber may be greater than the diameter of a lower chamber.
  • a high surface area fiber glass filter e.g., a borosilicate glass
  • the filter may be one that is adapted to isolate nucleic acids from a liquid sample by, for example, centrifugation, vacuum filtering, or any other filtering method.
  • Surface area refers to the total surface area of all the fibers and not just the area of the disk. The total surface area may range between 1,000 mm 2 and 50,000 mm 2 .
  • filter columns of the present invention may have a microliter capacity greater than 200 microliters. Another variation of the filter column having a microliter capacity ranging between 50 microliters and 1000 microliters. The microliter capacity is defined by the volume within a passageway of the filter column that above the filter.
  • Securing of the filter may be accomplished, for example, by placement of a disk of porous substrate material (not shown) at position 25.
  • a filter membrane (not shown) is placed on top of the substrate material.
  • a retaining ring (not shown) is added such that there is an interference between the retaining ring and the wall of the passageway. Accordingly, the retaining ring secures the filter on the porous substrate.
  • Another means of securing the filter is to mold an integral grating within the passageway to seat the filter.
  • Figure 3 illustrates placement of a filter using the porous substrate and retaining ring.
  • FIG. 3 illustrates a cut-away profile view of a preferred embodiment of a filter column 1 of the present invention.
  • the illustration shows a filter 29 held in the lower portion of the filter column 1.
  • the filter 29 is held between a porous support membrane 31, which has the same diameter as the interior of the lower portion of the spin filter 1.
  • the support membrane 31 is retained in place by the shoulder formed by the reduced diameter of the bottom 13 of the device.
  • the filter 29 may be fixed against the support membrane 31 by a retaining ring 33 which fits securely against the inner wall of the lower portion of the filter column 1.
  • the present invention is not limited to the previous illustration. It will be apparent to those skilled with previously known filter columns to provide other means of retaining the filter within a filter column.
  • An example of a filter for use with the invention includes a borosilicate glass fiber filter.
  • the device may also contain filters of other types such as polymeric membranes, and may also contain other functional groups for purification of the nucleic acid such as ion exchange groups or groups which would specifically bind nucleic acid sequences.
  • Another useful material for the porous substrate is a sintered polyethylene or polypropylene as supplied by Porex® of Fairburn, Georgia or GenPore® of Reading, Pennsylvania.
  • the porous substrate is comprised of a hydrophobic material in order to minimize holdup of the aqueous solution. It was found that pore sizes in the range of 10 micrometre to 150 micrometres worked well.
  • the primary function of the porous material is support for the filter. It is possible to have the porous support as integral to the device.
  • the porous support can also be in the form of an open grating.
  • Figure 4 illustrates a preferred embodiment of a filter column 1 of the present invention that is mated with a standard collection tube 35 of a first size (e.g., a 1.5 mL tube.)
  • Standard collection tubes contain an integral body made of polymers such as polypropylene and have a standard inner diameter.
  • the second body portion 5 of the filter column 1 may fit securely against an inner walls of the collection tube 35.
  • the first bearing surface 9 may rest against a shoulder formed by a rim 37 of the collection tube 35. Accordingly, when the tube 35 and filter column 1 spin in the centrifuge, liquid placed into the filter column 1 is forced through a filter 29 and collects in the collection tube 35.
  • the sizing of the first body portion 5 along with the bearing surface 9 permits stable placement of the filter column 1 within in the collection tube 35. After centrifugation, the filter column can be easily removed from the used collection tube, and placed into a new collection tube or placed back into the original tube.
  • Figure 5 illustrates the filter column 1 of Figure 4 placed within a collection tube 39 (e.g., a 0.5 mL tube) that is smaller than the tube illustrated in Figure 4 .
  • a collection tube 39 e.g., a 0.5 mL tube
  • the third body portion 7 of the filter column 1 fits securely into the inner walls of the collection tube 39.
  • a bearing surface 11 of the filter column 1 seats against a rim 41 of the collection tube 39.
  • FIG. 6A shows another preferred embodiment of a filter column 42 of the present invention having features which may be applied to any embodiment of the invention disclosed herein, within the scope of the appended claims.
  • the filter column 42 includes a lid 45 having a portion 47 which may be removably secured within a first end 49 of the filter column 42.
  • the lid 45 may assist in preventing contamination of the filter column 42 and any solutions loaded therein.
  • this embodiment of the filter column 42 contains a vent 27.
  • the present invention also contemplates a lid as being discrete from a filter column.
  • the preferred embodiment of the inventive filter column 42 also may include one or more snap-fit beads or ridges 53 to assist in retention of a lid 45.
  • the ridge 53 is included in the in a portion of a passageway in a first body portion 3 of the filter column 42.
  • the lid 45 and hinge 51 may be attached to the underside of an outer rim 55 ( e.g., subflush to the rim 55) of the filter column 42. This placement allows the outer rim 55 to be mated to other devices as required. (e.g ., a EtracSureTM sample extraction device useful for laser capture microdissection supplied by Arcturus Engineering of Mountain View, CA.)
  • deformable ribs 57 which are located along an outside surface of the filter column 42, as defined in appended claim 1.
  • the deformable ribs 57 assist in securing the filter column 42 in a slightly larger diameter tube (e.g., a 2.0 mL tube) by increasing a diameter of the filter column 42. Accordingly, these ribs deform upon insertion of the filter column 42 into a tube having a diameter slightly larger than the corresponding body portion upon which the ribs 57 are situated.
  • the number and design of the deformation ribs 57 may vary as needed, however, the ribs should be placed on a portion of the filter column that accommodates the varying sizes of tubes.
  • the invention also contemplates deformation ribs 57 which are either plastically or elastically deformable, or exhibit a limited degree of plastic or elastic deformation.
  • Figure 6B illustrates another preferred embodiment of the invention in which a filter column 59 contains at least one protrusion 61 which extends radially away from the filter column 59.
  • protrusions 61 may serve as "finger grips" to increase the ease with which the filter column 59 may be manipulated.
  • Figures 7-9 represent a flow diagram illustrating isolation of nucleic acids such as DNA or RNA from a biological material 80 using a filter column of the present invention.
  • Figure 7 represents the first step of placing a lysate 82 of biological material 80 in a collection tube 84. This lysate 82 is spun to remove large lytic debris, and the "cleared" lysate is then applied to the top of a filter column 86 of the present invention.
  • the nucleic acid containing solution does not have to come directly from lysis of biological materials.
  • This method can also purify nucleic acids following nucleic acid amplification, enzymatic restriction digestion, ligation, extension and virtually any solution containing significant nucleic acids.
  • nucleic acid solutions where small amounts of nucleic acid solutions are used, it is desirable to pre-wet the filter column with binding buffer before applying the nucleic acid solution.
  • Figure 8A illustrates the solution passing though a filter of the filter column 86 after centrifugation, during which the nucleic acids bind to the filter.
  • the flow-through solution (from which the nucleotides have been mostly extracted) is retained at the bottom of a collection tube 88.
  • tubes 82, 84 are discarded after use.
  • the binding buffer (flow through) is discarded, and the filter column is replaced in a collection tube (either a new tube or the same tube).
  • Figure 8B represents repeated washing of the filter column 86 with a washing buffer and centrifuging after each addition of washing solution into the top of the filter column. Once again the flow-through accumulated at the bottom of the tube 88 is discarded.
  • Figure 9 represents the last step of transferring the filter column 86 of the present invention into a smaller 0.5 mL tube 92 and the addition of a small volume of elution buffer into the filter column 86.
  • the inventive filter column permits use of more than one collection tube.
  • centrifugation steps can take place in a standard bench-top microcentrifuge (for example, Eppendorf 5415C), usually at accelerations less than 20,000g.
  • the biological material used with the filter column may also be provided by laser capture microdissection extraction device as described in U.S. patent application serial No. 09/844,187 , entitled "LASER CAPTURE MICRODISSECTION (LCM) EXTRACTION DEVICE AND DEVICE CARRIER, AND METHOD FOR POST-LCM FLUID PROCESSING.”
  • a filter column 1 attached to a LCM extraction device flange interface 94 which seats a laser capture microdissection extraction device 96.
  • the biological material will be located on a bottom surface of the extraction device 96.

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Claims (21)

  1. Colonne de filtration (1, 42) comprenant :
    un corps comportant une voie de passage (21) s'étendant à travers celui-ci, ledit corps comprenant au moins une première partie de corps (3), une deuxième partie de corps (5) et une troisième partie de corps (7), où un diamètre externe de ladite première partie de corps (3) est supérieur à un diamètre externe de ladite deuxième partie de corps (5), et où ledit diamètre externe de ladite deuxième partie de corps (5) est supérieur à un diamètre externe de ladite troisième partie de corps (7) ;
    un filtre (29) situé à l'intérieur de ladite voie de passage, ledit filtre (29) étant conçu pour isoler des acides nucléiques à partir d'un échantillon liquide ;
    une première surface d'appui (9) conçue pour reposer contre le contour (37) d'un premier tube de prélèvement (35) où un diamètre externe de la deuxième partie de corps (5) situé en dessous de la première surface d'appui (9) est conçu pour s'encastrer de manière sécurisée avec le premier tube de prélèvement (35), le premier tube de prélèvement (35) étant un tube centrifuge de 1,5 à 2,0 ml ; et
    une seconde surface d'appui (11) conçue pour reposer contre le contour (41) d'un second tube de prélèvement (39) où un diamètre externe de la troisième partie de corps (7) situé en dessous de la seconde surface d'appui (11) est adapté pour s'encastrer de manière sécurisée avec le second tube de prélèvement (39) et l'ouverture du premier tube de prélèvement étant supérieure à l'ouverture du second tube de prélèvement (39), le second tube de prélèvement (39) étant un tube centrifuge de 0,5 ml ou de 0,2 ml, une première extrémité (49) dudit corps étant adaptée pour recevoir une interface de bride de dispositif d'extraction de microdissection par capture laser (94) et
    des côtes déformables (57) étant situées le long d'une surface extérieure d'une partie de corps (5, 7) de la colonne de filtration (1, 42) qui est conçue pour accommoder lesdits premier et second tubes de collecte (35, 39).
  2. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle un diamètre interne de ladite voie de passage (21) dans ladite première partie de corps (3) est supérieur à un diamètre interne de ladite voie de passage (21) dans ladite deuxième partie de corps (5).
  3. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle un diamètre interne de ladite voie de passage (21) dans ladite deuxième partie de corps (5) est supérieur à un diamètre interne de ladite voie de passage (21) dans ladite troisième partie de corps (7).
  4. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle ledit filtre (29) est situé à l'intérieur d'une partie de ladite voie de passage (21) dans ladite deuxième partie de corps (5).
  5. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle ledit filtre (29) est situé à l'intérieur d'une partie de ladite voie de passage (21) dans ladite troisième partie de corps (7).
  6. Colonne de filtration (1, 42) selon la revendication 1, comprenant en outre un couvercle (45) ayant une partie (47) conçue pour être reçue de manière amovible à l'intérieur d'une première extrémité (49) dudit corps.
  7. Colonne de filtration (1, 42) selon la revendication 6, dans laquelle ladite première partie de corps (3) comprend une lèvre annulaire (53) conçue pour sécuriser de manière amovible ladite partie de couvercle (47) dans une position fermée dans ladite première extrémité dudit corps.
  8. Colonne de filtration (1, 42) selon la revendication 6, comprenant en outre au moins une bouche d'air (27) disposée sur une paroi intérieure dudit corps et adjacente à ladite première extrémité (49) dudit corps, ladite bouche d'air (27) étant en communication fluidique avec un extérieur de ladite colonne de filtration (1, 42) pendant que ledit couvercle (45) est conçu pour être reçu de manière amovible à l'intérieur de ladite première extrémité (49) dudit corps.
  9. Colonne de filtration (1, 42) selon la revendication 8, dans laquelle ladite au moins une bouche d'air (27) comprend quatre bouches d'air.
  10. Colonne de filtration (1, 42) selon la revendication 6, dans laquelle ledit couvercle (45) comprend une charnière (51) faisant partie intégrale dudit corps.
  11. Colonne de filtration (1, 42) selon la revendication 10, dans laquelle ladite charnière (51) est décalée vers ladite première extrémité (49) dudit corps.
  12. Colonne de filtration (1, 42) selon la revendication 1, comprenant en outre une pluralité de protrusions (61) sur ledit corps et s'étendant de manière radiale en s'éloignant dudit corps.
  13. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle chacune desdites première et seconde surfaces d'appui (9, 11) est conçue pour recevoir des premier et second tubes de collecte (35, 39) respectifs de tailles différentes.
  14. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle ledit diamètre externe de ladite deuxième partie de corps (5) est compris entre 8,1 et 9,4 mm.
  15. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle ledit diamètre externe de ladite troisième partie de corps (7) est compris entre 6,4 et 6,9 mm.
  16. Colonne de filtration (1, 42) selon la revendication 1, comprenant en outre une quatrième partie de corps et une troisième surface d'appui située entre ladite troisième partie de corps et ladite quatrième partie de corps, un diamètre externe de ladite quatrième partie de corps étant inférieur à un diamètre externe de ladite quatrième partie de corps (7).
  17. Colonne de filtration (1, 42) selon la revendication 1, ayant une capacité de microlitre supérieure à 200 microlitres, ayant une surface de filtration supérieure à 1000 mm2 et ayant une hauteur depuis une partie supérieure dudit corps à une partie supérieure de ladite deuxième surface d'appui (11) étant inférieure à 12,7 mm
  18. Colonne de filtration (1, 42) selon la revendication 1, dans laquelle ladite interface de bride de dispositif d'extraction de microdissection par capture laser (94) est conçue pour recevoir un dispositif d'extraction par capture laser.
  19. Kit permettant d'isoler des acides nucléiques, le kit comprenant :
    une colonne de filtration (1, 42) conformément à l'une quelconque des revendications 1 à 18 ;
    au moins un tube de prélèvement (35, 39) ;
    un tampon de liaison contenant un agent chaotropique ;
    une solution de lavage ; et une solution d'élution.
  20. Procédé d'isolation d'une matière à base d'acides nucléiques consistant à : fournir une colonne de filtration (1,42) conformément à l'une quelconque des revendications 1 à 18 comprenant:
    l'installation de la colonne de filtration (1, 42) dans le premier tube de prélèvement (35) au niveau de la première surface d'appui (9) de telle sorte qu'un diamètre externe de la deuxième partie de corps situé en dessous de la première surface d'appui (9) s'encastre de manière sécurisée avec le premier tube de prélèvement (35) ;
    la fixation d'une interface de bride de dispositif d'extraction de microdissection par capture laser (94) qui reçoit un dispositif d'extraction de microdissection par capture laser (96) avec de la matière biologique située sur la surface inférieure ;
    le transfert de la solution contenant l'acide nucléique au filtre (29) de la colonne de filtration (1) en présence d'un agent capable de promouvoir la liaison de la matière à base d'acide nucléique au filtre (29) ;
    le transfert de la solution depuis la colonne de filtration (1, 42) vers le premier tube de prélèvement (35) par l'intermédiaire d'une force centrifuge ;
    le lavage de filtre de la colonne de filtration (29) avec une solution de lavage ;
    l'élimination de la colonne de filtration (1, 42) à partir du premier tube de prélèvement (35) ;
    la fourniture d'un deuxième tube de prélèvement (39), l'ouverture du deuxième tube de prélèvement (39) étant inférieure à l'ouverture du premier tube de prélèvement (35) ;
    le placement de la colonne de filtration (1, 42) dans le deuxième tube de prélèvement (39) au niveau de la seconde surface d'appui de telle sorte qu'un diamètre externe de la troisième partie de corps situé en dessous de la deuxième surface d'appui (11) s'encastre de manière sécurisée avec le second tube de prélèvement (39) ; et
    l'application d'un tampon d'élution et l'élution de la matière à base d'acides nucléiques depuis la colonne de filtration (1, 42) au moyen d'une force centrifuge.
  21. Procédé selon la revendication 20, dans lequel ladite solution contenant une matière à base d'acide nucléique consiste en une solution de lysat préparée à partir de matière biologique perturbée par une solution de perturbation.
EP02742051.2A 2001-06-15 2002-06-14 Dispositifs de colonnes de filtration et procedes de filtration associes Expired - Lifetime EP1399727B1 (fr)

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US882530 1986-07-07
US09/882,530 US7229595B2 (en) 2001-06-15 2001-06-15 Filtration column devices and methods of filtering therewith
PCT/US2002/018755 WO2002103325A2 (fr) 2001-06-15 2002-06-14 Dispositifs de colonnes de filtration et procedes de filtration associes

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CA2450866A1 (fr) 2002-12-27
US20020192656A1 (en) 2002-12-19
WO2002103325A3 (fr) 2003-07-03
EP1399727A2 (fr) 2004-03-24
WO2002103325A2 (fr) 2002-12-27

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